Serine ADPr on histones and PARP1 is a cellular target of ester-linked ubiquitylation.

ADP-ribosylation and ubiquitylation regulate various cellular processes, with the complexity of their interplay becoming increasingly clear, as illustrated by ADP-ribosylation-dependent ubiquitylation mediated by Legionella. Biochemical studies have reported ester-linked ubiquitylation of ADP-ribose by DELTEX ubiquitin ligases, yet the modification sites on cellular targets remain unknown. Here, our search for interactors of RNF114 revealed DNA-damage-induced serine mono-ADP-ribosylation as a cellular target for ester-linked ubiquitylation. By developing proteomics strategies tailored to the chemical features of this composite modification, combined with an enrichment method using the zfDi19 and ubiquitin interaction motif domain (ZUD) of RNF114 and specific chemical elution, we identified ADP-ribosyl-linked serine ubiquitylation sites in cells, including on histones and poly(ADP-ribose) polymerase 1. Engineering ZUD into a modular reagent enabled the detection of this dual modification by immunoblotting. We establish ADP-ribosyl-ubiquitylation as an endogenous serine post-translational modification and propose that our multifaceted, tailored methodology will uncover its widespread occurrence, along with other conjugation chemistries, across diverse signaling pathways.